While there is growing evidence for tumor-associated antigens in renal cell carcinoma (RCC), most spontaneous immune responses to the disease are ineffective at limiting tumor progression. One mechanism by which tumors promote their own progressive growth is by inducing or sensitizing T cells to apoptosis. We recently determined that the ability of several RCC cell lines to mediate significant levels of apoptosis in co-cultured T cells is largely abrogated when the co-incubations are performed either in the presence of neutralizing anti-TNF antibodies, or with ganglioside-depleted tumor cells. This suggestion of a synergistic effect between tumor-derived gangliosides and TNF for inducing T cell apoptosis is corroborated by studies with purified sources of TNF and the RCC-overexpressed ganglioside GM1, which demonstrate similar cooperativity for killing T cells when used together in vitro. Our previous work indicated that select gangliosides can inhibit NFkappaB activation in T cells, thereby sensitizing the lymphocytes to apoptosis by preventing their synthesis of NFkappaB-dependent, anti-apoptotic proteins. The mechanism by which gangliosides cause that defect, and the nature of the second signal actually inducing apoptosis, however, have remained undefined. Preliminary data now suggest that RCC-derived gangliosides and TNF synergize to induce T cell apoptosis by two different mechanisms. We hypothesize that tumor-derived gangliosides disrupt lipid rafts on T cell membranes, preventing effective NFkappaB activation through TNFR1, and hence rendering apoptosis the default pathway induced upon TNF binding. We also have evidence that TNF enhances the effectiveness with which RCC gangliosides induce proapoptotic effects on the mitochondrion, accelerating the onset and intensity of the ROS and MPT mediated by gangliosides alone. The experiments outlined in this proposal will both assess the importance of a TNF/ganglioside synergy to RCC-induced T cell apoptosis, and test our hypothesis that both of the proposed, synergistic mechanisms may be contributing to the T cell dysfunction in RCC.